Name: uptake of new lipid-coated nanoparticles containing falcarindiol by human mesenchymal stem cells
Uploaded: 2019-02-09T14:00:00
Description: Watch this Scientific Journal Video about Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells at JoVE.com

The authors thank Dr. Moustapha Kassem (Odense University Hospital, Denmark) for the human mesenchymal stem cells. The authors thank the Danish Medical Bioimaging Center for access to their microscopes. The authors thank the Carlsberg and Villum foundations for financial support (to E.A.C.). The authors acknowledge the financial support provided by the Niels Bohr Professorship award from the Danish National Research Foundation.

1. Nanoparticle synthesis by rapid solvent shifting technique
<ol>
	<li>Set up the following for the nanoparticles&#39; preparation: a block heater/sample concentrator, a desiccator, a digital dispensing system with a 1 mL glass syringe, a 12 mL glass vial, a magnetic stirrer, a magnetic flea (15 mm x 4.5 mm, in a cylindrical shape with polytetrafluoroethylene [PTFE] coating) inside the glass vial, and a rotatory evaporator.</li>
	<li>Dispense 2.4 mL of 250 &#181;M falcarindiol stock dissolved in 70% EtOH water mixture...

<ol>
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P., Al-Najami, I., Frett&#233;, X., Baatrup, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+polyacetylenes,+falcarinol+and+falcarindiol,+isolated+from+carrots+prevents+the+formation+of+neoplastic+lesions+in+the+colon+of+azoxymethane-induced+rats.">Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.</a> Food &amp; Function. 8, 964-974 (2017).</li><li>Hervella, P., Parra, E., Needham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+retention+of+chelated-copper+inside+hydrophobic+nanoparticles:+Liquid+cored+nanoparticles+show+better+retention+than+a+solid+core+formulation.">Encapsulation and retention of chelated-copper inside hydrophobic nanoparticles: Liquid cored nanoparticles show better retention than a solid core formulation.</a> European Journal of Pharmaceutics and Biopharmaceutics. 102, 64-76 (2016).</li><li>Hervella, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelation,+formulation,+encapsulation,+retention,+and+in+vivo+biodistribution+of+hydrophobic+nanoparticles+labelled+with+57Co-porphyrin:+Octyl+Amine+ensures+stable+chelation+of+cobalt+in+Liquid+Nanoparticles+that+accumulate+in+tumors.">Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Octyl Amine ensures stable chelation of cobalt in Liquid Nanoparticles that accumulate in tumors.</a> Journal of Controlled Release. , (2018).</li><li>Zhigaltsev, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-up+design+and+synthesis+of+limit+size+lipid+nanoparticle+systems+with+aqueous+and+triglyceride+cores+using+millisecond+microfluidic+mixing.">Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing.</a> Langmuir. 28 (7), 3633-3640 (2012).</li><li>Aubry, J., Ganachaud, F., Cohen Addad, J. -. 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A detailed protocol for fabricating lipid-coated nanoparticles for drug delivery with the simple, fast, reproducible, and scalable rapid injection method of solvent shifting was followed27,28 and is presented in this paper, as applied to falcarindiol. By controlling the speed of the injection of the organic phase into aqueous phase and by using coating lipids at appropriate concentrations to coat the falcarindiol core, particle in the sub-100 nm range could be ob...

Lipid-coated nanoparticles are seeing an increased interest regarding their function as drug delivery systems for cancer therapy1. Cancers have an altered lipid-metabolic reprogramming2 and an increased need for cholesterol to proliferate3. They overexpress LDLs1 and take in more LDLs than normal cells, to the extent that a cancer patient&#39;s LDL count can even go down4. LDL uptake promotes aggressive phenotypes5 resulting in proliferation and invasion in breast cancer6. An abundance of LDL receptors (LDLRs) is a prognostic indicator of metastatic potential7. Inspired by the LDL and its uptake by cancer cells, a new strategy has been called: Make the drug look like the cancer&#39;s food8. Thus, these new nanoparticle drug delivery designs8,9,10&#160;have been inspired by the core- and lipid-stabilized design of the natural LDLs11&#160;as a mechanism for delivering anticancer drugs to cancer cells. This passive targeting delivery system supports the encapsulating of, especially, hydrophobic drugs, which are usually given in oral dosage form but provide only a small amount of the drugs to the bloodstream, so limiting their expected efficacy12. As with the stealth liposomes13, a polyethylene glycol (PEG) coating helps to reduce any immunologic response and extends the circulation in the bloodstream for optimum tumor uptake by the purported enhanced permeation and retention (EPR) effect14,15. However, in addition to, in some instances, instability in the circulation and undesirable distribution in the system16, some obstacles remain unsolved, such as how and to what extent such nanoparticles are taken in by cells and what is their intracellular fate. It is here that this paper addresses the nanoparticle uptake of a particular hydrophobic anticancer drug falcarindiol, using confocal and epifluorescence imaging techniques.
The aim of the study is to fabricate lipid-coated nanoparticles of falcarindiol and to study their intracellular uptake in hMSCs.&#160;Thereby, potentially stabilizing its administration, overcoming the challenges associated with the delivery, and improving the bioavailability. Thus assessing a new delivery system for this anticancer drug. Previously, falcarindiol has been administrated orally via a high concentration purified falcarindiol as a food supplement17. However, there is need for a more structured approach to deliver this promising drug. Therefore, falcarindiol nanoparticles, with a phospholipid and cholesterol encapsulating monolayer with the purified drug constituting the core of the particle, were designed. The rapid injection method of solvent shifting, as recently developed by Needham&#160;et al.8, is used in this study to encapsulate the polyacetylene falcarindiol.
The method has previously been used for the fabrication of lipid nanoparticles to encapsulate diagnostic imaging agents18,19, as well as test molecules (triolein)27 and drugs (orlistat, niclosamide stearate)8,27,28. It is a relatively simple technique when carried out with the right molecules. It forms nanosized particles, at the limit of their critical nucleation (~20 nm diameter), of highly insoluble hydrophobic solutes dissolved in a polar solvent. The solvent exchange is&#160;accomplished by a rapid injection of the organic solution into an excess of antisolvent (usually, an aqueous phase in a 1:9 organic:aqueous volume ratio)20,21.
The compositional design of the nanoparticles give rise to multiple advantages. The DSPC:Chol components provide a very tight, almost impermeable, biocompatible, and biodegradable monolayer.The PEG provides a sterically stabilizing interface which acts as a shield from opsonization by the immune system, slowing any uptake by the reticuloendothelial system (liver and spleen) and protecting against the mononuclear phagocyte system, preventing their retention and degradation by the immune system, and hence, increasing their circulation half-time in blood22. This allows the particles to circulate until they extravasate at diseased sites, such as tumors, where the vascular system is leaky, allowing EPR-effect to give rise to passive accumulation of the particles. Additionally, the lipid coat&#160;allows one to have better control over the nanoparticles&#39; size by kinetically trapping the core at its critical nucleus dimension27,28. Lipids induce various surface properties (including peptide targeting, which was not yet available for this project), a pure drug core, and a low polydispersity22,27,28. The method used for particle size analysis is DLS, a technique that allows researchers to measure the size of a large number of particles at the same time. However, this method can bias the measurements to bigger sizes, if the nanoparticles are not monodispersed23. This issue is assessed with the lipid coat as well. More details of these fundamental designs and the quantification of all characteristics are given in other publications27,28.
The drug encapsulated in the nanoparticles is falcarindiol, a dietary polyacetylene found in plants from the Apiaceae family. It is a secondary metabolite from the aliphatic C17polyacetylenes type that has been found to display health-promoting effects, including anti-inflammatory activity, antibacterial effects, and cytotoxicity against a wide range of cancer cell lines. Its high reactivity is related to its ability to interact with different biomolecules, acting as a very reactive alkylating agent against mercapto and amino groups24. Falcarindiol has previously been shown to reduce the number of neoplastic lesions in the colon17,25, although the biological mechanisms are still unknown. However, it is thought that it interacts with biomolecules such as NF-&#954;B, COX1, COX-2, and cytokines, inhibiting their tumor progression and cell proliferation processes, leading to arresting the cell cycle, endoplasmic reticulum (ER) stress, and apoptosis17,26 in cancer cells. Falcarindiol is used in this study as an example anticancer drug due to&#160;its anticancer potential and mechanism are being studied currently, and because it shows promising anticancer effects. The cellular uptake of the nanoparticles is tested in hMSCs and imaged using epifluorescence and confocal microscopy. This cell type was chosen due to its large size, making them ideal for microscopy.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">12 mL Screw Neck Vial (Clear glass, 15-425 thread, 66 X 18.5 mm)</td>
			<td style="width:384px;">Microlab Aarhus A/S</td>
			<td style="width:160px;">ML 33154LP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6 well plates</td>
			<td>Greiner Bio One International GmbH</td>
			<td style="width:160px;">657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Absolute Ethanol</td>
			<td>EMD Millipore (VWR)</td>
			<td>EM8.18760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloroform</td>
			<td>Rathburn Chemicals Ltd.</td>
			<td>RH1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cholesterol</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Confocal Microscope</td>
			<td style="width:384px;">Zeiss LSM510</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cover Slips thickness #1.5</td>
			<td>Paul Marienfeld GmbH &#38; Co</td>
			<td>117650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Desiccator</td>
			<td style="width:384px;">Self-build</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DiI</td>
			<td>Invitrogen</td>
			<td style="width:160px;">D282</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">DLS</td>
			<td style="width:384px;">Beckman Coulter</td>
			<td style="width:160px;">DelsaMAXpro 3167-DMP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DSPC (Chloroform stock)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850365C&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DSPE PEG 2000 (Chloroform stock)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>880120C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">eVol XR</td>
			<td>SGE analytical science, Trajan Scientific Australia Pty Ltd.</td>
			<td>2910200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fetal Bovine serum</td>
			<td>Gibco</td>
			<td style="width:160px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fluorescence Miccroscope</td>
			<td style="width:384px;">Olymous IX81</td>
			<td style="width:160px;"></td>
			<td>With Manual TIRF and Andor iXon EMCCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Incubator</td>
			<td style="width:384px;">Panasonic&#160;</td>
			<td style="width:160px;">MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnetic flea</td>
			<td>VWR Chemicals</td>
			<td>15 x 4.5 mm</td>
			<td>Cylindrical shape with PTFE coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnetic stirrer</td>
			<td>IKA</td>
			<td style="width:160px;">RT-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media</td>
			<td>Gibco</td>
			<td style="width:160px;">32561-029</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">PBS tablets for cell culture</td>
			<td style="width:384px;">VWR Chemicals</td>
			<td>97062-732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pen/strep</td>
			<td>VWR Chemicals</td>
			<td>97063-708</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Phosphate Buffer Saline (PBS, pH 7.4)</td>
			<td>Thermo Fisher</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rotary Evaporator</td>
			<td>Rotavapor, B&#252;chi Labortechnik AG</td>
			<td style="width:160px;">R-210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sample concentrator&#160;</td>
			<td>Stuart, Cole-Parmer Instrument Company, LLC</td>
			<td style="width:160px;">SBHCONC/1</td>
			<td></td>
		</tr>
	</tbody>
</table>

uptake of new lipid-coated nanoparticles containing falcarindiol by human mesenchymal stem cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

Two different types of nanoparticles were fabricated, namely pure falcarindiol nanoparticles and lipid-coated falcarindiol nanoparticles. Various concentrations of lipids and cholesterol were tested. As shown in Table 1, uncoated nanoparticles formed in water and measured in PBS had a diameter of 71 &#177; 20.3 nm with a polydispersity index (PDI) of 0.571. Those parameters were measured on a DLS analyzer. The lipid-coated nanoparticles of falcarindiol used in the experim...

Watch this Scientific Journal Video about Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells at JoVE.com

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

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